Androgenic steroids reduce muscle atrophy through molecular mechanisms that are poorly understood. In studies of the effects of the androgenic steroid nandrolone on atrophy of muscle paralyzed by nerve transection, we have found that rates of atrophy are reduced within 7 days after beginning nandrolone when this steroid is started at 29 days after nerve transection (35 days) associated with reduced expression of the muscle ubiquitin ligases MAFbx and MuRF1. By contrast, when nandrolone was begun at the time of nerve transection, it did not slow atrophy or reduce expression of MAFbx or MuRF1 7 days later (7 days). We believe that this model provides a unique opportunity to understand the molecular mechanisms by which nandrolone slows muscle atrophy, and to understand why muscle is resistant to the beneficial effects of nandrolone under some conditions. Of interest, gene profiling revealed that, among genes affected by nandrolone at 35 but not 7 days, and potentially capable of regulating muscle size, were FOXO1 and RCAN2. RCAN2 is expressed at high levels in skeletal muscle, where it inhibits calcineurin, a protein phosphatase involved in muscle hypertrophy by activating the transcription factor NFAT. Of interest, FOXO1 overexpression also reduces calcineurin activity, possibly by upregulating MAFbx [5-7], suggesting that nandrolone-induced reductions in FOXO1 and MAFbx levels may represent a second, parallel mechanism by which nandrolone may increase calcineurin activity in denervated muscle. The insensitivity of denervated muscle to nandrolone at early time points most likely reflects expression of a transcriptional regulator that blocks nandrolone action, or downregulation of one that is necessary for it. We found large differences in expression levels of several transcriptional coregulators in denervated skeletal muscle at 7 versus 35 days after denervation, with the greatest change, a 25-fold decrease, being for Ankrd2. We propose that analysis of effects of these transcriptional coregulators on nandrolone sensitivity will provide new insights into molecular determinants of muscle to effects of nandrolone and other androgens and explain the intriguing problem of resistance of skeletal muscle to androgens under some pathophysiological conditions. Major Aims of this proposal are, in a rat model of denervation atrophy, to determine: 1) whether nandrolone increases calcineurin activity in denervated muscle, and whether such increases are due to nandrolone-dependent changes in levels of calcineurin, RCAN2, and upstream regulators of calcineurin levels (FOXO1 and MAFbx);2) whether specific transcriptional coregulators mediate resistance to effects of nandrolone to reduce atrophy and repress MAFbx. PUBLIC HEALTH RELEVANCE: Significance Restoration of function in those recovering from acute illness or injury, and preservation of function in those with chronic medical conditions to maintain independence are major goals in rehabilitation medicine. A significant impediment to rehabilitation is muscle atrophy as a consequence of paralysis, immobilization, medications, and chronic illness. Approaches to minimizing atrophy focus on treating the underlying disease causing muscle loss, on nutrition, and on various forms of physical conditioning. These approaches do not directly regulate the biological processes causing muscle loss. The development of safe and effective pharmaceuticals that promote muscle recovery, block muscle loss, or both would be a huge step forward in speeding up rehabilitation therapy and discharge from the hospital. The two major expected outcomes of our studies are greater insights into specific mechanisms by which androgens prevent muscle atrophy, and new information about the molecular determinants of such protection. In addition to expanding general knowledge, this information holds the potential to improve future therapeutics that safely prevent muscle atrophy. Relevance to the VHA Mission Muscle loss affects veterans with diverse medical conditions that include stroke, spinal cord injury, COPD, asthma and rheumatological disease. It also affects wounded soldiers during recovery from burns and/or immobilization resulting from their injuries. Knowledge that improves treatments to preserve or restore function of these veterans holds the potential to significantly improve the speed of their recovery, and, possibly, their quality of life and community function and integration.